A large number of diseases, such as cancer, are associated with genetic modifications, including strand breaks, DNA-adducts, and DNA-protein cross links. Thus, a number of prior art methods have been developed in order to detect and to quantitate modifications in DNA.
Prior art methods for the detection and quantitation of nucleic acid modifications include .sup.32 P-postlabeling [Randerth et al. (1981) Proc. Natl. Acad. Sci. USA 78:6126-6129; Gupta et al. (1996) In Technologies for Detection of DNA Damage and Mutations, G.P. Pfeifer (ed.) Plenum, New York, pp. 45-61], Gas chromatography in combination with mass spectrometry (GC/MS) [Dizdaroglu (1993) FEBS 315:1-6; Niritsin and Markey (1996) Anal. Biochem. 241:35-41], and high-performance liquid chromatography (HPLC) in combination with electrochemical and mass spectrometry detection [Wagner et al. (1992) Proc. Natl. Acad. Sci. 78:3380-3384]. However, these methods suffer from several drawbacks, including poor sensitivity. In addition, these methods also involve a series of chemical derivatization and/or enzymatic hydrolysis and labeling steps which can introduce artifactual DNA lesions, and which require that digestion and labeling reactions be optimized.
Enzyme-linked immunosorbent assays (ELISA) have also been used to detect DNA lesions [Leadon and Hanawlat (1983) Mutat. Res. 112:191-200; Cooper et al. (1997) Science 275:990-993; Melamede et al. (1996) In Technologies for Detection of DNA Damage and Mutations, G.P. Pfeifer (ed.) Plenum, New York, pp. 103-115]. However, these assays require large amounts of starting material (microgram quantities of DNA), and are time consuming. Importantly, all the above-disucussed methods have low sensitivity, and are useful only for detecting greater than femtomole (10.sup.-15 mole) levels of DNA lesions.
Yet other methods, such as pulse-field gel electrophoresis and single-cell gel electrophoresis, have been employed to detect and measure nucleic acid sequence mutations. While these methods are sensitive, their use is limited to the detection and measurement of strand breaks.
To date, the art has attempted to circumvent the low sensitivity of available methods for detecting and measuring DNA modifications by exposing cells or whole organisms (e.g., rodents) to ionizing radiation or to carcinogenic chemicals at doses which are significantly greater than those doses encountered in the environment or in clinical settings (e.g., clinical ionizing radiation), followed by extrapolating back from the dose-response curves in order to postulate on the effect of environmentally and clinically relevant doses. This approach, however, lacks reliability since the effects of treatment with low and high doses of carcinogens have disparate effects both on DNA lesion formation and on DNA lesion repair.
Thus, what is needed are methods for detecting and measuring modifications of nucleic acid sequences. Preferably, these methods should be sensitive, specific, use small amounts of nucleic acid sequences, and should not require the use of hazardous radioactive compounds, of enzymatic digestion or of chemical derivatization of the nucleic acid substrate.